Interactions of Arg2 in the Mnt N-terminal arm with the central and flanking regions of the mnt operator

Arg2 in the N-terminal arm of the Mnt repressor, plays an important role in determining operator-binding specificity. Ln the complex of the Mnt tetram er with the 21 base-pair mnt operator, there are four potential sites for A rg2 interactions, two in the central region of the operator and two on the outer flanks of the operator. Single-chain variants of the dimeric N-termin al domain of Mnt containing one Arg2 residue and one Lys2 or Met2 residue w ere constructed and interactions with operator DNA were probed using Fe EDT A affinity cleavage. The results of these orientation studies show that the majority of the energetically significant interactions mediated by Arg2 oc cur in the central region of the mnt operator. The RK2, RA2, and RM2 mutati ons reduce the free energy of operator binding by 1.7 kcal/mol, 3.3 kcal/mo l, and 4.9 kcal/mol, respectively. Double-mutant thermodynamic cycle analys es using the RA2, RM2, and operator variants also reveal interaction free e nergies between Arg2 and operator base-pairs 9, 10, 11, 12 and 13, which in aggregate account for most of the Arg2 contribution to operator binding. ( C) 2000 Academic Press.